1. Field of the Invention
The present invention relates generally to the field of stem cell development. More particularly, it concerns the generation of pluripotent stem cells.
2. Description of Related Art
The unlimited proliferation capability and pluripotent potential of human embryonic stem (ES) cells have offered unprecedented access to all cell types of the human body. Human induced pluripotent stem (iPS) cells derived directly from patient somatic cells with desired genetic background share these two key properties of human ES cells, which made these cells excellent candidates for disease models, drug screening, toxicity testing and transplantation therapies. Initial derivation of human iPS cells employed genome-integrating retroviral or lentiviral vectors to deliver reprogramming transgenes (Lowry et al., 2008; Park et al., 2008; Takahashi et al., 2007; Yu et al., 2007). Such vectors can produce insertional mutations that interfere with the normal functions of human iPS cells and their derivatives, and residual transgene expression that can influence differentiation into specific lineages (Yu et al., 2007), or even result in tumorigenesis (Okita et al., 2007).
iPS cells free of exogenous genetic elements have been derived from mouse embryonic fibroblasts with repeated plasmid transfections (Okita et al., 2008), from mouse liver cells and human fibroblasts with non-integrating adenoviral vectors (Stadtfeld et al., 2008; Zhou and Freed, 2009), from somatic cells with piggyback transposons (Woltjen et al., 2009), from human fibroblasts with oriP/EBNA-1-based episomal vectors (Yu et al., 2009) and protein transduction. Despite these rapid advances, major hurdles remain to prevent the wide use of any single technology that produce high-quality human iPS cells free of exogenous genetic elements. For example, all the current technologies (except the piggyback transposon approach) that allow generation of human iPS cells free of exogenous genetic elements yielded very low reprogramming efficiency. This low efficiency makes it difficult to obtain iPS cells consistently from a variety of easily accessible human somatic cell types, and from cells with different genetic background and donor age. The piggyback transposon approach offers reasonable reprogramming efficiency. However, the removal of transposons from iPS cells can be quite labor-intensive when many donor cell lines are involved.
In addition, despite the high similarity of human iPS cells to human ES cells, there exist significant clone-to-clone variations both in gene expression/epigenetic modifications and in the lineage-specific differentiation potential of human iPS cells. In particular, compared to human ES cells, most human iPS cells exhibit significantly lower neural differentiation potential and no response to LIF (leukemia inhibitory factor), which routinely supports mouse ES cell culture. Moreover, due to the lack of good easily assayable markers for high-quality human iPS cells, selection of high-quality human iPS cell clone can be labor-intensive and time-consuming.
Genetic reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs) could offer replenishable cell sources for transplantation therapies. To fulfill their promise, human iPSCs will ideally be derived and cultured in chemically defined media free of feeder cells, and be free of exogenous DNA (footprint-free). Currently, there is not a simple and efficient feeder-free nonviral method for the generation of footprint-free human iPSCs. Previously efforts of footprint-free human iPSCs by employing episomal vectors for transgene delivery were inefficient and required feeder cells.
Therefore, there remains a need to address the inefficiency or other problems in preparing induced pluripotent stem cells essentially free of exogenous genetic components.